β-arrestin1 protects intestinal tight junction through promoting mitofusin 2 transcription to drive parkin-dependent mitophagy in colitis

Abstract Background Intestinal barrier defect is an essential inflammatory bowel disease (IBD) pathogenesis. Mitochondrial dysfunction results in energy deficiency and oxidative stress, which contribute to the pathogenesis of IBD. β-arrestin1 (ARRB1) is a negative regulator that promotes G protein-coupled receptors desensitization, endocytosis, and degradation. However, its role in maintaining the intestinal barrier remains unclear. Methods Dextran sulfate sodium-induced colitis was performed in ARRB1 knockout and wild-type mice. Intestinal permeability and tight junction proteins were measured to evaluate the intestinal barrier. Mitochondria function and mitophagic flux in mice and cell lines were detected. Finally, the interaction between ARRB1 and mitofusin 2 was investigated by co-immunoprecipitation and dual luciferase assay. Results We identified that ARRB1 protected the intestinal tight junction barrier against experimental colitis in vivo. ARRB1 deficiency was accompanied by abnormal mitochondrial morphology, lower adenosine triphosphate (ATP) production, and severe oxidative stress. In vitro, the knockdown of ARRB1 reduced ATP levels and mitochondrial membrane potential while increasing reactive oxygen species levels and oxidative stress. Upon ARRB1 ablation, mitophagy was inhibited, accompanied by decreased LC3BII, phosphatase and tension homologue-induced protein kinase1 (PINK1), and parkin, but increased p62 expression. Mitophagy inhibition via PINK1 siRNA or mitochondrial division inhibitor 1 impaired ARRB1-mediated tight junction protection. The interaction of ARRB1 with E2F1 activated mitophagy by enhancing the transcription of mitofusin 2. Conclusions Our results suggest that ARRB1 is critical to maintaining the intestinal tight junction barrier by promoting mitophagy. These results reveal a novel link between ARRB1 and the intestinal tight junction barrier, which provides theoretical support for colitis treatment.


Introduction
Inflammatory bowel disease (IBD), including Crohn's disease (CD) and ulcerative colitis (UC), is a multifactorial disease involving immune dysregulation, genetic susceptibility, environmental factors, and microbial dysbiosis [1].The intestinal epithelial barrier defends against para-cellular permeation of endogenous and exogenous noxious antigens and pathogens in the intestinal lumen.Accumulating evidence indicates that the loss of intestinal barrier integrity contributes to increased intestinal permeability, triggering or accelerating intestinal inflammation [2,3].The intestinal barrier comprises several cellular components, including tight junctions, adherence junctions, and desmosomes [4].Tight junctions limit the para-cellular permeability of luminal proinflammatory molecules [5].Increased intestinal permeability has been observed in IBD, accompanied by abnormal tight junctions structure and a down-regulation of several tight junctions proteins, including claudins and occludin [6].Recent research has demonstrated that regulation of tight junctions integrity ameliorates intestinal inflammation [7,8].Thus, protecting epithelial barrier function could be a potential therapeutic strategy for IBD.
Oxidative stress is essential in the pathogenesis of gastrointestinal mucosal diseases [9].Mitochondria are the primary source of intracellular reactive oxygen species (ROS).Since maintaining intestinal epithelial cells junction integrity is energy-dependent, mitochondrial function might be central to the appropriate preservation of epithelial barrier function.It has been shown that the potential factors that induce mitochondrial dysfunction contribute to IBD susceptibility [10,11].Mitochondrial abnormal structure and dysfunction have been observed in patients with IBD [12], and mitochondrial ROS and mitochondrial DNA from damaged mitochondria are proinflammatory factors [13].Mitophagy is a necessary and specific form of autophagy that selectively removes dysfunctional or redundant mitochondria [14].Mitophagy has been demonstrated to have a neuroprotection effect in Alzheimer's disease [15,16] and be involved in innate immunity [17,18].Compromised mitophagy has further been observed in functional studies of CD-associated risk variants in autophagy-related ATG16L1 and immunity-related GTPase M [19].Preliminary studies indicate mitophagy as a protective process in IBD by promoting proinflammatory cytokine production, intestinal epithelial cell viability, and possibly pathogen clearance [20,21].Furthermore, mitophagy has become integral to immune cell development, activation, and differentiation [22].Thus, maintaining mitophagy might play a uniquely protective role in preventing the disease progression of IBD.
β-arrestin1 (ARRB1) is a negative G protein-coupled receptor (GPCR) signaling regulator.It promotes GPCR desensitization, endocytosis, and degradation [23].ARRB1 has also been demonstrated as a molecular scaffold that regulates cellular function through interactions with other partner proteins and contributes to immune response, inflammation, and tumorigenesis [24].Mounting evidence also suggests that ARRB1 can translocate from the cytoplasm to the nucleus and modulate targeted gene transcription [25].Nuclear ARRB1 interacts with transcription cofactors, such as p300 and IκBα, to play a vital role in cell growth, apoptosis, and immune function [26,27].ARRB1 has been identified as a critical regulator of glucose and energy homeostasis [28].Pan et al. [29] found that high glucose attenuated the cardioprotective effects of glucagon-like peptide 1 through induction of mitochondrial dysfunction via inhibition of ARRB1.Nevertheless, ARRB1 has increased mitochondrial oxidative stress in cultured human cardiac fibroblasts [30].Hence, the role of ARRB1 in mitochondrial function requires further investigation.
The role of ARRB1 in IBD is still controversial.A previous study found the deficiency of ARRB1 protects against experimental colitis [31,32].However, we previously demonstrated that ARRB1 mediated mucosal protection by COX-1/PGE2/EP4 in colitis [33].Furthermore, it has not yet been established whether ARRB1 regulates epithelial barrier function in colitis.Our study demonstrated that ARRB1 deficiency impaired the intestinal tight junctions barrier through mitochondrial dysfunction accompanied by lower adenosine triphosphate (ATP) production and increased oxidative stress in colitis mice.Mechanistically, ARRB1 enhanced mitophagy to maintain mitochondrial function through activating mitofusin 2 (MFN2) transcription.These results reveal a novel link between ARRB1 and the intestinal tight junction barrier in colitis, which provides theoretical support for colitis treatment.

Mice
All animal procedures were approved by the Research Ethics Committee of the Third Affiliated Hospital of Sun Yat-Sen University (IACUC-F3-20-0911).C57BL/6 genetic background mice were used.The original Arrb1 heterozygous mice were kindly provided by Dr R. J. Lefkowitz from Duke University Medical Center (Durham, NC, USA).All colonies were housed in micro isolator cages with 50% humidity and 12-h light-dark cycles.Six-to eight-week-old and sex-matched mice were randomly assigned to groups.Three percent dextran sulfate sodium (DSS; MP Biomedicals LLC, Solon, OH, USA) in drinking water for 7 days was administered to induce acute colitis in mice.
pLenti-ARRB1-puromycin (GeneChem, Shanghai, China) was used for lentiviral transfection to generate the stable ARRB1overexpression cell line.ARRB1-shRNA was cloned into the lentiviral vector GV112 (GeneChem) to generate the stable ARRB1 knockdown cell line.Stable transfections were selected with respective antibiotics for 2 weeks.

Plasmid transfection
Briefly, 70%-80% of confluent cells were transfected.Lipofectamine 3000 reagent (Invitrogen, Waltham, MA, USA) was used to deliver plasmid DNAs into cells growing in serum-free opti-MEM media.Subsequent experiments were completed 24 h after transfection.The GFP-LC3 and parkin plasmids were kindly provided by Professor Yunfei Qin (Department of The Biological Therapy Center, The Third Affiliated Hospital of Sun Yat-Sen University, Guangzhou, China).ARRB1 plasmid was kindly provided by Dr Gan Pei (Chinese Academy of Sciences, Shanghai, China).PINK1, MFN2, E2F1, ARRB1 (Q394L), and ARRB1 (1-163) plasmids were created and synthesized by YouminBio (Guangzhou, Guangdong, China).The pcDNA3.0-Vector was transfected as the negative control.

Quantitative RT-PCR
Total RNA was extracted using TRIzol (No. 15596-018; Invitrogen) and transcribed into cDNA using a High Capacity cDNA Kit (No. FSQ101; TOYOBO, Japan).Then aliquots of cDNA were amplified using gene-specific primers and ChamQ SYBR qPCR Master Mix (No. Q441; Vazyme, Nanjing, Jiangsu, China) in a real-time PCR system (Bio-Rad, Hercules, CA, USA).Each sample was tested in triplicate.Relative expression levels were calculated by the 2 −ΔΔCt method and normalized to β-actin.The sequences of the primers are listed in Supplementary Table 1.

Western-blot analysis and co-immunoprecipitation
Total protein was either isolated from the intestinal tissues or cells using a lysis buffer supplemented with a protease and phosphatase inhibitor cocktail (No. 78430; Thermo Scientific, Waltham, MA, USA).Immunoblotting was performed as described previously [33].Western blotting was performed using antibodies against ARRB1 (1:1000; No. ab32099; Abcam), SQSTM1/p62 (1:1000; No. .Polyvinylidene fluoride membranes with proteins were incubated overnight with primary antibodies at 4 � C and corresponding secondary antibodies at room temperature for 2 h.Proteins were visualized using a Bio-Rad ECL machine.The protein bands were quantified using ImageJ software (US National Institutes of Health, Bethesda, MD, USA).
For immunoprecipitation, cells were lysed in IP Lysis Buffer (No. P0013; Beyotime) with a protease and phosphatase inhibitor cocktail (No. 78430; Thermo Scientific).The lysate was precleared with protein A magnetic beads (No. 1614013; Bio-Rad) at 4 � C overnight and then incubated with anti-ARRB1 and anti-E2F1 antibodies for 2 h.Antimouse or rabbit IgG antibodies (No.A7016/No.A7028; Beyotime) from the related species were used as a control.After removing the beads by centrifugation, the boiled samples were subjected to immunoblot analysis.

Cell viability assay
Cell counting kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan) assays were used to measure cell viability.HCoEpic cells (2.5 × 10 3 /well) were seeded into 96-well plates.After treatment with the indicated chemicals, 10 μL CCK-8/90 μL culture medium was added to each well.After incubation at 37 � C for 2 h, the absorbance at 450 nm was measured by a spectrophotometer (BioTek-Epoch2; Santa Clara, CA, USA).The experiments were performed in triplicate wells and three times independently.

ATP measurement
The ATP content was measured using the ATP assay kit (No. S0027; Beyotime).Cells or tissue samples were washed with icecold PBS and then homogenized and sonicated in lysis buffer on ice.After sonication, the lysed cells or tissues were centrifuged at 12,000×g for 5 min to remove debris.Then, ATP was determined using the ATP assay kit based on the luciferin/luciferase assay and normalized for protein content.

Measurement of malondialdehyde and glutathione
The malondialdehyde and glutathione levels were measured according to the manufacturer's protocol (Boxbio Science & Technology, Beijing, China).Malondialdehyde measurement was determined by the reaction of thiobarbituric acid (TBA) with malondialdehyde to generate the stable end product of the malondialdehyde-TBA adduct.The cells were lysed by sonication, and the tissue samples were homogenized on ice.Then the samples were centrifuged at 8,000×g for 10 min at 4 � C, and the supernatants were mixed with the malondialdehyde detection working solution and incubated at 100 � C for 60 min.After cooling to room temperature, the mixtures were centrifuged at 10,000×g for 10 min, and the supernatants were evaluated using a spectrophotometer (BioTek-Epoch2) at 450, 532, and 600 nm wavelengths.The malondialdehyde content was calculated by the difference of the value at 450, 532, and 600 nm.
For glutathione measurement, the samples were centrifuged at 10,000×g for 10 min at 4 � C, and the supernatants were subjected to the glutathione assay kit and mixed with the glutathione detection working solution.Then the output was measured immediately at 412 nm by a spectrophotometer (BioTek-Epoch2).The protein concentration of each sample was determined by a bicinchoninic acid protein assay kit (No. 23227; Thermofisher).In addition, malondialdehyde and glutathione levels were normalized according to the protein concentrations.

Mitochondrial membrane potential
Mitochondrial membrane potential (MMP) was detected with JC-1 staining.When the membrane potential is low, JC-1, as a monomer, emits green excitation light.At higher membrane potentials, JC-1 aggregates increase and emit red light.After treatment with the indicated drugs, cells were incubated with 5 mg/mL JC-1 (No. C2006; Beyotime) for 20 min at 37 � C, avoiding light and washed twice with PBS.Then, a fluorescence microscope (Zeiss, Oberkchen, Germany) or FACS flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) measured the red and green fluorescence cell ratio.

Scanning and transmission electron microscopy
Fresh colon tissues were washed with cold PBS and fixed with ice-cold 2.5% glutaral overnight.Fixed colon tissues were cut into blocks of 1 mm 3 thickness.Tissues were fixed with 1% osmium tetroxide for 1 h at room temperature, followed by dehydration using graded ethanol.Tissues were embedded in epoxy resin overnight and sectioned into 100 nm slices.Electron microscopy samples were obtained at the electron microscopy core lab of Sun Yat-Sen University with the scan and transmission electron microscope (JEOL, Japan).

Intestinal and transepithelial permeability
Intestinal and transepithelial permeability were detected by FITC-dextran (4000 MW; FD4; Sigma Aldrich) as previously described [34].Mice were administered 0.6 mg/g body weight FD4 in PBS by oral gavage.Blood was collected 4 h later by retro-orbital bleeding.The serum concentration of FITC-dextran was determined using a microplate reader (Infinite 200 pro; TECAN) with an excitation wavelength of 490 nm and an emission wavelength of 530 nm.Serial-diluted FITC-dextran was used to generate a standard curve.The transepithelial permeability was assessed by apical to basolateral FD4 transmission in the transwell plate (Corning 3460; Corning, USA).Briefly, 5 mg/mL FD4 was added to the up-chamber, and after several hours, the sample of the down-chamber was detected by the microplate reader.The transepithelial permeability was presented as the concentration of FD4 in the basolateral chamber.

MitoTracker staining
After treatment, cells were incubated with 200 nM MitoTracker Red CMXRos (No. C1035; Beyotime) for 30 min at 37 � C in the dark.Then the cells were washed with PBS and observed using a laser scanning confocal microscope (Leica, Germany) and a microplate reader.

Mitochondrial isolation
Mitochondria were extracted using the mitochondrial extraction kit (No. C3601; Beyotime).Cells were rinsed in pre-cold PBS and homogenized in 1 mL ice-cold lysis buffer with a pre-cool Dounce-type glass homogenizer.Then the homogenate was centrifuged 3 times (1000×g for 10 min at 4 � C) to pellet cell debris and nuclei, and collected the supernatants.Finally, mitochondria from the supernatant were pelleted by centrifugation at 12,000×g for 10 min at 4 � C.

Quantification of mitochondrial DNA
Total cellular DNA was extracted using the DNeasy Blood and Tissue kit (No. 51011; Bio-Generating, Changzhou, Jiangsu, China) and quantified using a NanoDrop2000 spectrophotometer (BioTek-Epoch2).A total of 100 ng DNA was amplified using ChamQ SYBR qPCR Master Mix (Vazyme) in a real-time PCR system (Bio-Rad).The target gene content was normalized to β-globin DNA.The primers are listed in Supplementary Table 1.

Dual-luciferase reporter assay
The Dual-Luciferase Reporter Assay System was used to measure luciferase activity.Transfection efficiency was normalized to Renilla luciferase activity.

Statistical analysis
Data were presented as means±SD.The statistical significance was analyzed using Student's t-tests or one-way analysis of variance tests (ANOVA), and all tests were two-tailed.The Pearson correlation coefficient was used to estimate the correlation between the mRNA expression levels of MFN2 and ARRB1.The statistical significance was set at P < 0.05.

ARRB1 upregulates intestinal barrier function by protecting tight junction during inflammation
Consistent with our previous study [33], H&E staining showed that DSS induced more colonic mucosal injury in ARRB1 knockout (KO) mice (Figure 1A).Next, we addressed whether the susceptibility of ARRB1 deficiency to colitis was due to an intestinal epithelial barrier defect.Intestinal permeability was evaluated by the FD4 test.The serum concentration of FD4 in experimental colitis was significantly higher in ARRB1 KO mice than in wild-type (WT) mice, indicating that ARRB1 deficiency contributed to increased intestinal permeability (Figure 1B).The integral membrane components of tight junction proteins regulate the selective permeability between epithelial cells.According to scanning electron microscopy, the tight junction between cells on the apical side of the colonic epithelium was significantly more damaged in ARRB1 KO mice than in WT mice with colitis (Figure 1C).The mRNA of occludin and claudin 1, two principal components of tight junctions, were reduced more in ARRB1 KO mice compared with WT mice.The mRNA of ZO-1 was the same reduced whether ARRB1 deficiency or not (Figure 1D).Immunofluorescence staining showed that claudin 1 and occludin were markedly decreased in ARRB1 KO mice than in WT mice (Figure 1E).This result was confirmed by Western-blot analysis (Figure 1F).These results indicated that ARRB1 deficiency impaired the integrity of the intestinal tight junction barrier during colitis.Afterward, we further investigated the potential protective effect of ARRB1 on the tight junction barrier function of HCoEpic cells.Compared with the negative control (Vector), ARRB1 overexpression (Lv-ARRB1) increased the mRNA levels of claudin 1 and occludin but not ZO-1 (Figure 1G).ARRB1 promoted claudin 1 and occludin expression in Western blotting (Figure 1H).In the presence of TNF-α, the concentration of FD4 in the Lv-ARRB1 group increased at a much slower rate, indicating a protective role of ARRB1 on trans-epithelial permeability (Figure 1I).Together, ARRB1 protects the intestinal barrier

ARRB1 deficiency exacerbates mitochondrial dysfunction and oxidative stress in the colonic epithelium of experimental colitis
As indicated by swelling of the mitochondria, fracture of the inner or outer membranes, and rupture of the mitochondrial crest, transmission electron microscopy showed that the mitochondria of intestinal epithelial cells from colitis in ARRB1 KO mice were more severely damaged than those of WT mice in colitis (Figure 2A).Then we analyzed the number, length, and width of mitochondria in a double-blinded manner.There were fewer mitochondria and a higher ratio of round mitochondria (length/ width <2) in ARRB1 KO mice (Figure 2B and C).These results indicated that the ARRB1 deficiency resulted in a decreased number and severe morphological abnormalities of mitochondria.COX IV, a mitochondrial marker, was reduced more in ARRB1 KO mice (Figure 2D).The mRNA expressions of mitochondrial cytochrome c oxidase II (MT-CO2) and mitochondrial cytochrome B (MT-CYB), mitochondrial DNA encoded-mitochondrial complexes subunits, were remarkably reduced in ARRB1 KO mice (Figure 2E).These results indicated that colitis induced more mitochondrial damage in ARRB1 KO mice.Next, we investigated the effects of ARRB1 on mitochondrial function.In ARRB1 KO mice, colitis induced significant exhaustion of glutathione and elevation of malondialdehyde, which indicated a deficit in antioxidant capacity (Figure 2F and G).Similarly, ATP production in the colon tissues of ARRB1 KO mice was significantly lower than that in WT mice (Figure 2H).

Mitochondrial dysfunction-induced ROS mediates down-regulation of tight junctions in vitro
It is unknown whether mitochondrial dysfunction disrupts the intestinal tight junction.
Next, we investigated the effect of mitochondrial function on tight junctions in HCoEpic cells.We used CCCP, an uncoupler of oxidative phosphorylation, as an inducer of mitochondrial dysfunction [35].We detected the mitochondrial function and tight junction at different times to explore changes in CCCP-induced mitochondrial dysfunction over time.We found a decrease in the viability of the cells after CCCP treatment for 24h (Figure 3A).Reduced ATP and glutathione levels appeared after CCCP treatment for 12h (Figure 3B and C).The protein expressions of claudin 1 and occludin were reduced after CCCP treatment for 12h (Figure 3D and E).Cellular permeability was also increased by FD4 measurement (Figure 3F).Those results showed that tight junction and cellular permeability were damaged simultaneously with mitochondrial dysfunction but earlier than the decline of cell viability.Mitochondria are the primary source of cellular ROS.NAC, a ROS scavenging agent, significantly reduced cellular ROS accumulation following CCCP treatment (Figure 3G).NAC did not rescue the decreased viability induced by CCCP (Figure 3H).The CCCPinduced downexpression of claudin1 and occludin was reversed by NAC, indicating that ROS was responsible for reducing these proteins induced by mitochondrial dysfunction (Figure 3I and J).

Knockdown of ARRB1 reduces MMP, antioxidant activity, and ATP production in vitro
To clarify the effect of ARRB1 on mitochondrial function In vitro, we assessed MMP by measuring the ratio between red and green fluorescence by JC-1 staining.The knockdown of ARRB1 resulted in a more pronounced reduction of MMP in response to TNF-α exposure (Figure 4A and B).Sh-ARRB1 cells consistently exhibited faint red and intense green fluorescence under a fluorescence microscope (Figure 4C).Moreover, the knockdown of ARRB1 increased cellular ROS accumulation (Figure 4D).As mitochondrial DNA copy numbers can predict the relative number of mitochondria, we extracted it exclusively from the cells and selected MT-CO2 and MT-CYB to represent mitochondrial DNA.We found that TNF-α decreased the levels of MT-CO2 and MT-CYB, but ARRB1 was resistant to this reduction (Figure 4E).ARRB1 delayed TNF-α-induced mitochondrial quantity reduction as assessed by Mito-Tracker Red CMXRos probe staining (Figure 4F and G).The knockdown of ARRB1 reduced antioxidant activity with a significant decrease in glutathione and an evaluation of malondialdehyde level (Figure 4H and I).The production of ATP was also reduced in sh-ARRB1 cells (Figure 4J).Thus, these results suggest that ARRB1 knockdown reduces MMP and disrupts mitochondrial function in vitro.

ARRB1 preserves mitochondrial function through promoting PINK1/parkin-dependent mitophagy
Mitophagy defects lead to damaged mitochondria accumulation and pathological change.PINK1/Parkin-mediated mitophagy is a significant pathway of mitophagy.Having demonstrated that the absence of ARRB1 disrupted mitochondrial function, we hypothesized that ARRB1 preserved mitochondrial quantity and function through PINK1/parkin-mediated mitophagy.Transmission electron microscopy showed an increase in mitochondrial autophagosomes in DSS-induced colitis.ARRB1 KO mice exhibited fewer mitochondrial auto-phagosomes than WT mice (Figure 5A).Western blotting showed that the mitophagyassociated proteins PINK1, Parkin, and LC3B were significantly upregulated in experimental colitis, but ARRB1 KO inhibited the upregulation of these proteins (Figure 5B and C).These results demonstrated that the deletion of ARRB1 inhibited mitophagy, which is activated to clear damaged mitochondria in colitis.Next, we examined the effect of ARRB1 on the mitophagic flux in HCoEpic cells.The co-localization of LC3 with COX IV, an indicative mitophagy marker, was less prominent in sh-ARRB1 cells than in sh-NC cells (Figure 5D).We analyzed the colocalization of LC3 and mitotracker.CQ treatment increased the colocalization of LC3 and mitotracker because CQ blocks autophagosome degradation.We found that mitophagy was rescued after transfecting parkin to ARRB1 KO cells (Figure 5E).ARRB1 knockdown significantly downregulated the mRNA expressions of PINK1 and Parkin (Figure 5F).TNF-α increased the expressions of PINK1, Parkin, and LC3B-II.However, the knockdown of ARRB1 suppressed these up-expressions (Figure 5G).We isolated mitochondria to confirm that these mitophagy-associated proteins were changed specifically in mitochondria.TNF-α enriched PINK1, Parkin, and LC3B-II in the mitochondrial fraction, but this effect was suppressed in sh-ARRB1 cells (Figure 5H).Taken together, ARRB1 promotes PINK1/parkin-mediated mitophagy.

Inhibition of mitophagy downregulates ARRB1-mediated tight junction protection
Subsequently, we validated the importance of dysregulated mitophagy on intestinal tight junctions by ARRB1 knockdown.The silencing of ARRB1 downregulated the expression of claudin 1 and occludin, and NAC reversed this downexpression (Figure 6A).Thus, the reduction of tight junction proteins by ARRB1 knockdown was due to ROS accumulation.Then, we used PINK1 siRNA to downregulate expressions of PINK1.In addition, the downregulation of PINK1, Parkin, and LC3B-II indicated that si-PINK1 inhibited mitophagy, undermining ARRB1-mediated claudin 1 and occludin up-regulations (Figure 6B and C).Additionally, transfecting with PINK1 plasmid to sh-ARRB1 cells, we found that claudin 1 and occludin expressions were restored (Figure 6D and E).Moreover, we used Mdivi-1 as a mitophagy inhibitor.Mdivi-1 inhibited mitophagy and reduced the expressions of claudin 1 and occludin (Figure 6F-H).Furthermore, the inhibition of mitophagy medicated by si-PINK1 and Mdivi-1 significantly reduced glutathione levels (Figure 6I).

ARRB1 protects tight junction through MFN2-mediated mitophagy
Next, we intended to investigate the mechanisms by which ARRB1 modulated mitophagy.MFN2 located on the outer mitochondrial membrane, is not only the regulator of mitochondrial fusion but also involved in the progression of mitophagy.By analyzing two GEO data (GSE87466 and GSE 107499), we discovered that MFN2 was significantly down-regulated in UC (Figure 7A).Then, we found that silencing MFN2 with siRNA reduced ATP production and glutathione levels, indicating that MFN2 deficiency contributed to mitochondrial dysfunction (Figure 7B  and C).
We analyzed GSE87466 and GSE107499 data and revealed a significant positive correlation between MFN2 and ARRB1 mRNA (Figure 7D).Thus, we hypothesized that ARRB1 regulated mitophagy via MFN2.We demonstrated that the knockdown of ARRB1 reduced MFN2 mRNA and protein expression (Figure 7E and F).Subsequently, we detected MFN2 expression in colonic tissue.MFN2 expression was reduced in colitis, and ARRB1 deficiency exacerbated this decrease (Figure 7G).Immunohistochemistry presented consistent results (Figure 7H).

Interaction of ARRB1 with E2F1 enhances MFN2 transcription
We then investigated how ARRB1 regulated MFN2 expression.It is known that ARRB1 translocates from the cytoplasm to the nucleus, interacting with transcription co-factors such as p300 and NF-kappaB to modulate targeted gene transcription [26,27].A previous study demonstrated that E2F1 activates MFN2 expression by binding to its promoter [36].Consistent with this finding, we found that the knockdown of E2F1 inhibited ARRB1-mediated MFN2 upexpression (Figure 8A), and the transfection of E2F1 restored the repression of MFN2 by the knockdown of ARRB1 (Figure 8B).Co-immunoprecipitation showed an interaction between ARRB1 and E2F1 (Figure 8C and D).These results indicated that ARRB1 might regulate MFN2 expression by interaction with E2F1.However, E2F1 worked as a transcriptional factor in the nucleus.To identify whether nuclear translocation of ARRB1 regulates the expression of MFN2, we performed transient transfection with ARRB1 mutant Q394L, in which glutamine 394 has been mutated to leucine to create a nuclear export signal [37].The ARRB1 mutant Q394L failed to rescue MFN2 expression in sh-ARRB1 cells, suggesting that nuclear translocation of ARRB1 was required for transcriptional regulation of MFN2 (Figure 8E and F).The Luciferase activity assay also showed that the mutant Q394L did not enhance E2F1-mediated transcription from the MFN2 promoter (Figure 8G).Previous studies have shown that amino acids 1-163 of ARRB1 are required to bind to E2F1 [38].Thus, ARRB1 1-163 could competitively inhibit the interaction of ARRB1 WT and E2F1.The luciferase activity assay showed that ARRB1 1-163 inhibited the ARRB1-mediated MFN2 upexpression by competitive binding to E2F1 (Figure 8G).Consistent with the luciferase activity assay, ARRB1 WT increased the MFN2 mRNA levels, but not ARRB1 mutant Q394L.Moreover, the ARRB1mediated upregulation of MFN2 was inhibited by ARRB1 1-163 (Figure 8H).As a result, ARRB1-E2F1 interaction enhanced MFN2 transcription.

Discussion
ARRB1 was initially discovered and described as a negative regulator of G protein-dependent signaling in the GPCR signaling pathway [23].Further research has found that ARRB1, as a scaffolding protein, interacts with several effector proteins [39].ARRB1 regulates fundamental biological processes such as proliferation and apoptosis [40].Our previous study found that ARRB1 is involved in COX-1/PGE2/EP4-mediated mucosal protection in colitis [33].However, the effect of ARRB1 on colitis remains unclear.Lee et al. [31] initially found that the deficiency of ARRB1 protects against experimental colitis, then found that nonhematopoietic ARRB1 confers protection against experimental colitis [32].ARRB1-dependent signaling in hematopoietic and nonhematopoietic cells differentially regulates colitis pathogenesis.Due to the contribution of intestinal epithelial barrier impairment to the initiation and progression of IBD, we sought to investigate the role of ARRB1 in the intestinal barrier.ARRB1 deficiency reduced the expression of claudin 1 and occludin, which are essential components of the intestinal tight junctions barrier.ARRB1 protects the tight junction of the intestinal epithelium to maintain the function of the intestinal barrier.As a crucial component of the physical barrier, the tight junction between intestinal epithelial cells regulates paracellular permeability.Emerge evidence in vivo and In vitro has shown decreased expression of tight junctions and increased intestinal permeability due to reduced epithelial barrier function [41].Moreover, maintaining the integrity and function of the intestinal epithelial barrier may contribute to preventing and treating IBD [42].Therefore, ARRB1 is critical in maintaining intestinal tight junction barrier function.
Recent evidence has placed mitochondria as the gatekeeper of intestinal epithelial cell homeostasis [43].Mitochondria supply energy and essential metabolites for cell activities.It has been shown that dysregulated mitochondrial signaling and function contribute to the pathogenesis of IBD [44].Two recent studies proved that Paneth cells are highly susceptible to mitochondrial dysfunction in IBD.Prohibitin, a substantial component protein of the inner mitochondrial membrane, is crucial for the optimal assembly and function of the respiratory chain.Mice lacking prohibitin in intestinal epithelial cells developed Paneth cell abnormalities and spontaneous ileitis preceded by mitochondrial dysfunction [45].In another study, Khaloian et al. [46] demonstrated that inflammation-associated mitochondrial dysfunction in the intestinal epithelium triggered a metabolic imbalance and drove intestinal stem cells to transition into aberrant Paneth cells.Zhang et al. [47] found that knockdown of ARRB1 inhibited isoproterenol-induced mitochondrial ROS production.High glucose induces mitochondrial dysfunction by inhibiting ARRB signaling to attenuate the cardio-protective effects of glucagon-like peptide 1 [29].However, some studies have found the opposite result.The deletion of ARRB1 did not affect cerebral ischemia-induced inflammation and oxidative stress but markedly suppressed autophagy and induced neuronal apoptosis/ necrosis [48].
Recent research has demonstrated the association between ARRB1 and autophagy.ARRB1 regulates BECN-dependent autophagosome formation to mediate neuroprotection in cerebral ischemia [48].Lei et al. [49] indicated that HBx-induced hepatocellular carcinogenesis through ARRB1-mediated autophagy.Mitophagy is protective in eliminating dysfunctional or redundant mitochondria to maintain mitochondrial homeostasis [50].Mitophagy impairment perturbs mitochondrial function and causes progressive accumulation of defective organelles, leading to cell and tissue damage [51].Mitophagy plays a vital role in intestinal inflammation.Vincent et al. [52] found that NIX deficiency aggravated colitis via mitophagic inhibition, which induced the inability to clear damaged or dysfunctional mitochondria.Consistently, we found mitophagy was involved in ARRB1-mediated tight junction protection.ARRB1 deficiency suppressed PINK1-parkin-dependent mitophagy and induced severe oxidative stress and ROS accumulation.Mitofusins are preferred targets at the OMM and are ubiquitylated by E3 ligases [53].MFN2 is a regulator of mitochondrial fusion and is associated with mitophagy.Upon mitophagy induction, ubiquitination of MFN2 targets them for degradation by proteasomes, quickly leading to the abrogation of mitochondrial fusion events and resulting in mitochondrial fragmentation.Thus, MFN2 acts as a pro-mitophagic receptor.It has been demonstrated that loss of MFN2 is associated with defects in autophagosome or autophagosome-lysosome formation, two events of mandatory nature for mitophagy [54].Consistently, depletion of MFN2 in murine cardiomyocytes caused an accumulation of defective mitochondria.MFN2 deficiency inhibited mitophagy and increased apoptosis [55].MFN2-induced mitophagy improved disease prognosis in gastric cancer [56].In this work, we found that ARRB1 inhibited mitophagy through decreasing MFN2 expression.Down-expression of MFN2 leads to suppressing PINK1/parkindependent mitophagy, subsequently hindering the elimination of dysfunctional mitochondria.Furthermore, ARRB1 regulates MFN2 transcriptionally.ARRB1 is a scaffold protein in multiple signaling pathways and co-factors gene transcription regulation.Zecchini et al. [57] demonstrated that nuclear ARRB1-induced pseudohypoxia and cellular metabolism reprogramming in prostate cancer via regulation of HIFA transcription activity.Another study indicated that ARRB1 is bound to E2F1 target genes that modulate epithelial-mesenchymal transition in nicotine-induced growth of lung tumors [37].This study demonstrated that ARRB1 regulates MFN2 transcription by cooperating with E2F1.This finding explains how ARRB1 functions as a transcriptional regulator in cell growth, apoptosis, and mitochondrial function modulation.
In conclusion, the KO of ARRB1 resulted in mitochondrial dysfunction and a disrupted intestinal barrier in colitis.Mechanistically, ARRB1 promoted PINK1/parkin-mediated mitophagy to eliminate damaged mitochondria.Silencing of ARRB1 resulted in ROS accumulation and exacerbated oxidative stress.As a transcriptional regulator, ARRB1 interacted with E2F1 to promote MFN2 transcription and enhance cell mitophagy flux.
Our findings may open new therapeutic avenues for maintaining mitochondrial dysfunction to treat colitis.
β-arrestin1 protects intestinal tight junction by mitophagy | 5 function by upregulating the expression of tight junction proteins during inflammation.